Project Summary The epidermal growth factor receptor (EGFR) tyrosine kinase is one of the most commonly activated oncoproteins in lung adenocarcinoma, glioblastoma and other cancers. The identification of cancer-associated EGFR mutants and their predictive power to select patients for treatment with EGFR inhibitors constitutes a major advance in treatment of these cancers. The collaborative efforts of the Eck and Meyerson laboratories over the past 12 years have provided a detailed structural and mechanistic understanding of selected activating and resistance mutations in EGFR, and have led to new classes of inhibitors that can overcome treatment-acquired resistance. In this renewal, we propose to perform studies that will inform the discovery of inhibitors of EGFR variants for which current treatments are ineffective, including exon20 insertion mutants in non-small cell lung cancer and EGFRvIII mutants in glioblastoma. In addition, we propose to probe the structure and regulation of the intact receptor to better understand its oncogenic activation. We will undertake the following Specific Aims: Aim 1) Analyze the biochemical and structural impact of small-molecule kinase inhibitors on EGFR exon20 insertion mutants, Aim 2) Develop allosteric inhibitors that selectively inhibit the EGFRvIII variant in glioblastoma, and Aim 3) Structure elucidation of the complete EGF receptor. Execution of these aims will lead to small molecules that will serve as templates for next-generation therapeutics for EGFR- driven cancers that do not respond to current EGFR targeted agents. Additionally, our structural and biochemical studies will illuminate mechanisms of receptor activation and signaling in both normal and oncogenic contexts that may suggest new avenues for therapeutic intervention in the future.